JPS6344532A - Vaccine for reovirus infection disease or swine - Google Patents

Abstract

PURPOSE:To obtain the titled vaccine, by proliferating reovirus of swine in a culture cell and inactivating the virus. CONSTITUTION:Swine reovirus separated from swine affected by respiratory disease is inoculated in SK cell (swine kidney primary culture cell) and cultured at 37 deg.C for 5-8 days. Collected virus liquid is added with an inactivation agent (e.g. 0.15vol% formalin), sensitized at about 22 deg.C for about 48hr and added with 20vol% aluminum phosphate gel or 50-70vol% hydrophilic oily adjuvant to obtain the objective vaccine for reovirus infection disease of swine. The vaccine has excellent effectivity and safety to respiratory disease of swine caused by infection with said virus and is extremely useful to prevent the serious damage on the management of swinery.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This invention relates to a vaccine for swine reovirus infection.

[Conventional technology]

Porcine reovirus in Europe and the United States was successively isolated from the respiratory or gastrointestinal tract around 1970, and its pathogenicity was confirmed by infection tests using piglets that had not received colostrum, leading to lesions mainly in the lungs accompanied by respiratory symptoms. It has been shown that it causes gastrointestinal lesions accompanied by diarrhea. In Japan, there have been no reports on the isolation of reovirus from pigs, and the inventors of this invention were the first to successfully isolate it in 1984 at two farms in Niigata and Yamagata prefectures. A factor that exhibits a cytopathic effect on primary cultured pig kidney (SK) cells was isolated from the nasal swabs and tonsils of slightly weakened pigs with enucleation or rhinorrhea in a fattening pig group, and the various properties of this isolated factor were investigated. As a result, it was identified as reovirus.

When such isolated reoviruses are inoculated orally or nasally to piglets that have not received colostrum, they exhibit respiratory symptoms such as mild enucleation, sneezing, and rhinorrhea, as well as soft stools or watery diarrhea, and histopathologically, Degeneration of the nasal mucosa or tracheal epithelium, swelling of the alveolar epithelium, and clustering of macrophages or lymphocytes are observed, and degeneration of epithelial cells and clustering of lymphocytes or macrophages are observed in the intestinal tract.

Until now, reovirus has not been isolated from pigs in Japan, so the damage caused by reovirus infection in pig herds in the field has not been clarified.However, since 1977, the inventors of this invention have We have been conducting disease surveys at large-scale pig farms. According to the results, respiratory diseases such as enucleation and rhinorrhea rapidly increased after the introduction of fattening pigs in introduced fattening pig farms and at the age of 3 to 4 months in conventional pig farms. A tendency for neutralizing antibodies to increase has also been confirmed. In the end, this virus infection in pigs overlaps with secondary bacterial infection and causes respiratory symptoms accompanied by various symptoms, and shows a decrease in energy and appetite, gradually weakening and causing pigs to be excluded from fattening or die. The amount of wear and tear caused by reovirus infection in the world is by no means small. 1
Based on the results of the antibody investigation conducted by the inventors of this invention in large-scale pig farms using preserved serum since 1977, the number of neutralizing antibodies against reovirus varies depending on the pig farm or pig group;
The positive rate is approximately 20% at ~2 months of age, but increases to approximately 50% at 3 to 4 months of age, and approximately 90% at 6 to 9 months of age.
It became clear that there was a tendency for the percentage to rise even further. As described above, the damage caused by reovirus infection in pigs tends to increase as the size of pig farms increases.

[Invention (Problem to be solved 3) As mentioned above, although reovirus infection in pigs tends to cause greater damage to the management of pig farms, conventional technology has no effective method to combat this infection. The problem was that a vaccine had not yet been developed.

[Means for solving problems]

In order to solve the above-mentioned problems, the present invention adopts a method of propagating Taku's reovirus in cultured cells and using an inactivated antigen as an active ingredient to create a vaccine. The details will be described below.

First, porcine reovirus is isolated and collected from a pig with respiratory disease, and this virus is inoculated into SK cells (the above-mentioned primary cultured pig kidney cells), cultured at 37°C for 5 to 8 days, and the virus is isolated. Collect the liquid.

The present invention can be achieved by adding 0.15% by volume of an inactivating agent such as formalin, sensitizing it at around 22°C for about 48 hours, and then adding 20% by volume of aluminum phosphate gel or 50 to 70% by volume of a hydrophilic oily adjuvant. swine reovirus infection vaccine can be obtained.

〔Example〕

Reovirus NNFOγ43 strain was inoculated into SK cells on the 5th day of culture, and after culturing at 37°C for 7 days, 0.15% by volume of formalin was added and sensitized at 22°C for 48 hours to inoculate the virus. was inactivated. An inactivated vaccine to which 20% by volume of aluminum phosphate gel was added and an inactivated vaccine to which 70% by volume of a hydrophilic oil-based adjuvant were added were prepared.

When the two types of inactivated vaccines obtained were injected twice, 2 ml each at two-week intervals, subcutaneously or intramuscularly into pigs, 8 to 128 times more neutralizing antibodies were produced two weeks after the second injection. Immunity lasted for 6 months, and no decrease in efficacy was observed even when the vaccine was stored in a cool, dark place at 2-5'C for 12 months.

The following results were obtained regarding the safety and effectiveness of these vaccines. In other words, safety: The first
As shown in the table, the aluminum phosphate gel-added vaccine and the hydrophilic oil-based adjuvant-added vaccine were each administered at
Subcutaneously and intramuscularly to the head, 2 weeks Table 1 Note 2 Vaccine injection amount is 2 ml, 2 times every 2 weeks
The animals were injected once, and two animals were kept together as untreated controls. We observed the reactions and clinical symptoms at the time of vaccination, and found that no abnormalities were observed in either the injected pigs or control pigs. Measurement of the antibody titer revealed that immunity lasted for at least 6 months.

Efficacy: 10 antibody-negative pigs approximately 2.5 months old were given 2 ml of the aluminum gel-added vaccine and the hydrophilic oil-adjuvanted vaccine subcutaneously and intramuscularly at 2-week intervals as shown in Table 2. In the 1st month after injection, reovirus NN
3 ml of 10'°6TCI D5°7ml of FOγ43 strain
ml After intranasal challenge, virus was recovered from nasal swabs every day for 14 days. Virus recovery in the vaccinated group was significantly lower than in the untreated challenge control group.
The effectiveness of this vaccine has been confirmed.

Furthermore, this vaccine was administered intraperitoneally to 4-week-old mice at 0, 5
ml was inoculated and observed for 7 days, but no abnormalities were observed and the body weight did not decrease.Furthermore, 4-week-old mice were intraperitoneally inoculated with 0.25 ml twice at 1-week intervals.
The serum collected from Higuchi's blood produced four times more neutralizing antibodies. 2ml each for 2-3 month old pigs that are negative for neutralizing antibodies.
Even after two subcutaneous or intramuscular injections at 3-week intervals, the pigs showed no abnormalities, and 8 to 128 times more neutralizing antibodies were produced 2 to 3 weeks after the second injection, indicating that the pigs had been immunized by vaccination. Even when challenged with live virus, such as intranasal inoculation, the amount of virus recovered from the nasal cavity was significantly lower than in control pigs, and it was confirmed that the pigs were protected against infection.

〔effect〕

As mentioned above, the swine reovirus infection vaccine of the present invention has excellent efficacy and safety for pigs against respiratory diseases caused by this virus infection, and has excellent efficacy and safety for pigs. This invention can be said to be of great significance since it greatly contributes to preventing serious damage to business management.

Claims (1)

[Claims] A porcine reovirus infection vaccine characterized by propagating porcine reovirus in cultured cells and inactivating it.